Synthesis, structure and thermodynamic properties of 8-methylguanine-containing oligonucleotides: Z-DNA under physiological salt conditions
Synthesis, structure and thermodynamic properties of 8-methylguanine-containing oligonucleotides: Z-DNA under physiological salt conditions
Hiroshi
Sugiyama
,
Kiyohiko
Kawai
,
Atsushi
Matsunaga
,
Kenzo
Fujimoto
,
Isao
Saito
,
Howard
Robinson
and
Andrew H.-J.
Wang
1,
*
Department of Synthetic Chemistry and Biological Chemistry, Faculty of
Engineering, Kyoto University,
Kyoto
606-01,
Japan
and
1
Department of Cell and Structural Biology, 506 Morrill Hall, University of
Illinois at Urbana-Champaign,
Urbana
, IL 61801,
USA
Received December 11, 1995
;
Revised and Accepted February 16, 1996
Brookhaven Protein Databank identifiers ITNE and RITNEMR
ABSTRACT
Various oligonucleotides containing 8-methylguanine (m
8
G) have been synthesized and their structures and thermodynamic properties
investigated. Introduction of m
8
G into DNA sequences markedly stabilizes the Z conformation under low salt
conditions. The hexamer d(CGC[m
8
G]CG)
2
exhibits a CD spectrum characteristic of the Z conformation under physiological
salt conditions. The NOE-restrained refinement unequivocally demonstrated that d(CGC[m
8
G]CG)
2
adopts a Z structure with all guanines in the
syn
conformation. The refined NMR structure is very similar to the Z form crystal
structure of d(CGCGCG)
2
, with a root mean square deviation of 0.6 between the two structures. The
contribution of m
8
G to the stabilization of Z-DNA has been estimated from the mid-point NaCl concentrations for the B-Z transition of various m
8
G-containing oligomers. The presence of m
8
G in d(CGC[m
8
G]CG)
2
stabilizes the Z conformation by at least
[Delta]
G = -0.8 kcal/mol relative to the unmodified hexamer. The Z conformation was
further stabilized by increasing the number of m
8
Gs incorporated and destabilized by incorporating
syn
-A or
syn
-T, found respectively in the (A,T)-containing alternating and non-alternating pyrimidine-purine sequences. The results suggest that the
chemically less reactive m
8
G base is a useful agent for studying molecular interactions of Z-DNA or other DNA structures that incorporate
syn
-G conformation.
INTRODUCTION
It has been well established that DNA structure has a remarkable conformational
heterogeneity (
1
,
2
). Not only does the biologically relevant B-DNA exhibit considerable local heterogeneity, dramatically different DNA
structures such as Z-DNA have also been discovered. While the precise biological functions of Z-DNA have yet to be identified, its role in regulating DNA
supercoiling has been amply demonstrated (
3
,
4
). A recent study by Rich and colleagues has shown that chicken double-stranded RNA adenosine deaminase has strong Z-DNA binding properties (
5
). This enzyme is known to work near the transcription apparatus, where a high
negative supercoiling density along the DNA chain exists in front of the site
of polymerase action (
2
). Thus far most of the thermodynamic properties of Z-DNA have been obtained through the use of supercoiled DNA plasmids
containing various alternating (C[middot]G)
n
inserts or their variants (
2
-
4
). However, other aspects of Z-DNA have not been thoroughly investigated, presumably due to the
difficulty of obtaining stable Z form oligonucleotides in a physiological salt
solution. Much of the available experimental data are limited to d(C[middot]G)
n
oligomers under non-physiological conditions of high alcohol or high salt concentrations (
6
-
8
). While some chemical modifications, such as C
5
-methylation or C
5
-bromination of cytosine (
9
) or C
8
-bromination of guanine (
10
,
11
), have been shown to stabilize the Z conformation in linear DNA oligomers, they
have either limited power for inducing the B-Z transition or they are chemically unstable. Therefore, it is desirable
to have a more convenient and reliable way to stabilize Z form oligomers under
low salt conditions by incorporating chemically and photochemically inert
modified bases. We report herein that the introduction of a methyl group at the
guanine C
8
position produces a stable m
8
-modified guanine base and markedly stabilizes the Z conformation of short
oligonucleotides of a variety of sequences under physiological salt conditions.
MATERIALS AND METHODS
Pyridine and acetonitrile (HPLC grade) were dried over calcium hydride. 2'-Deoxyguanosine (Yamasa Co.), nucleoside [beta]-cyanoethylphosphoramidite reagents (Applied Biosystems),
calf intestine alkaline phosphatase (AP) (1000 U/ml) and snake venom phosphodiesterase (s.v. PDE) (3 U/ml; Boehringer Mannheim) were all of the
highest grade. Silica gel columns and thin layer chromatography were carried
out on Wakogel C-200 and Merck silica gel 60 PF
254
plates respectively. FAB mass spectra were obtained in a JEOL-JMS-SX102A.
Synthesis of m
8
G-containing oligonucleotides
Introduction of a methyl group at the C
8
position of guanine was performed by the free radical methylation method (
12
). To a solution of
N
-isobutyryl-2'-deoxyguanosine (1.0 g, 2.97 mmol) and FeSO
4
[middot]7H
2
O (6.7 g, 24.1 mmol) in 160 ml 1 N H
2
SO
4
was added an aqueous solution (100 ml) containing 2.6 ml 70%
t
-butyl hydroperoxide (19.0 mmol) dropwise over a period of 5 min. After
stirring at 0oC for 60 min the reaction mixture was neutralized with saturated KOH
solution. The supernatant, obtained by centrifugation of the brown slushy
mixture, was concentrated to dryness and the resulting brownish solid was
triturated three times with 200 ml methanol. The combined methanol solution was
concentrated and the residue was subjected to silica gel column chromatography.
Elution with CH
2
Cl
2
/methanol (9:1) afforded 8-methyl-
N
-isobutyryl-2'-deoxyguanosine (
1
) as a white powder: yield 527 mg (51%), analytical data, m.p. 195oC (dec.);
1
H NMR (D
2
O, 200 MHz) [delta] 1.21 [d, 6 H, J = 6.9 Hz, -CH(C
H
3
)
2
], 2.25 (ddd, 1 H, J = 13.7, 6.9, 3.6 Hz, 2'), 2.56 (s, 3 H, -8CH
3
), 2.72 [sep, 1 H, J = 6.9 Hz, -C
H
(CH
3
)
2
], 3.17 (ddd, 1 H, J = 13.7, 7.4, 6.9 Hz, 2'), 3.72 (dd, 1 H, J = 11.9, 5.4 Hz, 5'), 3.74 (dd, 1 H, J = 11.9, 4.1 Hz, 5'), 3.92 (ddd, 1 H, 5.4, 4.1, 3.6 Hz, 4'), 4.59 (ddd, 1 H, J = 6.9, 3.6, 3.6 Hz, 3'), 6.32 (dd, 1 H, J = 7.4, 6.9 Hz, 1'); FABMS (positive ion)
m
/
z
352 (M+H)
+
.
1
was dimethoxytritylated according to a standard procedure to DMTr-
1
, which was further converted to its [beta]-cyanoethylphosphoramidite with the following analytical data:
1
H NMR (CD
3
OD, 200 MHz) [delta] 1.17 [d, 12 H, J = 6.5 Hz, -NCH(C
H
3
)
2
], 1.26 [dd, 6 H, J = 6.8, 3.4 Hz, -CH(C
H
3
)
2
], 2.41-2.64 [m, 3 H, 2', -NC
H
(CH
3
)
2
], 2.57 (s, 3 H, -8CH
3
), 2.63-2.77 [m, 1 H, -C
H
(CH
3
)
2
], 2.69 (t, 1 H, J = 5.9, -OCH
2
-), 2.84 (t, 1 H, J = 5.9, -OCH
2
-), 3.28-3.54 (m, 3 H, 2', -CH
2
CN), 3.72 (s, 3 H, -OCH
3
), 3.73 (s, 3 H, -OCH
3
), 3.54-3.71 (m, 2 H, 5'), 4.07-4.34 (m, 1 H, 4'), 4.09 (ddd, 1 H, J = 7.2, 3.4, 3.2 Hz, 4'), 4.64-4.83 (m, 1 H, 3'), 6.36 (t, 1 H, J = 7.3 Hz, 1'), 6.60-6.80 (m, 4 H,
aromatic), 7.06-7.44 (m, 9 H, aromatic);
31
P NMR (CD
3
OD, 80 MHz) [delta] 148.48; FABMS (positive ion)
m
/
z
854 (M+H)
+
. A set of m
8
G-containing DNA oligomers (
2-7
) (see Table
3
) were prepared by means of an automated DNA synthesizer. After deprotection
with ammonium hydroxide oligonucleotides were purified by HPLC and the
composition of the nucleosides was confirmed by enzymatic digestion.
NMR analysis
Analysis of the thermodynamic data
Circular dichroism (CD) spectra were recorded on a Jasco J-700 spectrophotometer equipped with a Peltier temperature controller. CD
spectra of oligonuclotide solutions (0.1 mM duplex in 30 mM phosphate, pH 7.0)
were recorded using a 1 cm path length cell. CD spectra at different
temperatures were recorded at intervals of 5oC with a 1 min equilibration period.
RESULTS AND DISCUSSION
Although theoretical calculations suggested that methylation at the guanine C
8
position greatly stabilizes the Z conformation by favoring the
syn
glycosyl conformation (
17
), such a property associated with m
8
G-modified DNA has not been examined experimentally. While introduction of
the bulky bromine atom at the C
8
position has been used previously (
10
,
11
), the brominated DNA suffered the problem of chemical/photochemical
instability. It would be desirable to use the more stable m
8
G in DNA to investigate the molecular basis of a variety of Z conformation-specific reactions at the oligonucleotide level.
The CD spectra of d(CGC[m
8
G]CG)
2
(
2
) at different salt concentrations are shown in Figure
1
A at 10oC. The hexamer in a 50 mM NaCl solution has the characteristic CD spectrum
of Z-DNA. Without added salt it is in the SS form, as judged by UV and CD
spectroscopy, and is converted to the Z form by increasing salt concentration,
with a mid-point at 4.5 mM NaCl. Since the respective mid-point NaCl concentrations for d(CGCGCG)
2
and d(m
5
CGCGm
5
CG)
2
are 2.6 M (
18
) and 2.0 M (
7
), it is evident that C
8
-methylation of guanine greatly stabilizes the Z conformation.
NMR refinement of Z-DNA
In order to unequivocally demonstrate that the structure of d(CGC[m
8
G]CG)
2
(
2
) at 30 mM salt concentration is Z-DNA, NOE-restrained refinement has been carried out. 2D NOESY and TOCSY in D
2
O were used to assign the resonances of all non-exchangeable protons. Since the structure is expected to be Z-DNA, as judged from the CD spectrum, the usual sequential assignment procedure would not be applicable. Indeed, the aromatic-H
1'
and m
8
G
4
methyl-H
1'
cross-peak region of the 2D NOESY spectrum (Fig.
2
) showed only strong intranucleotide G
2
H
1'
-G
2
H
8
, G
4
H
1'
-G
4
Me and G
6
H
1'
-G
6
H
8
cross-peaks, indicative of the
syn
conformation of guanine residues. As has been noted before (
6
,
19
), there is no internucleotide connectivity in Z-DNA, in contrast to that in right-handed B-DNA. The assignment was subsequently extended to the aromatic-H
2'
/H
2''
region and finally to all regions of the spectrum. The TOCSY data supported the assignment
(data not shown).
Thermodynamic parameters for Z-B transition of d(CGC[m
8
G]CG)
2
(
2
) and d(CGCGCG)
2
at 2.6 M NaCl
a
Oligonucleotide
[Delta]G
297K
[Delta]H
[Delta]S
(kcal mol
-1
)
(kcal mol
-1
)
(eu)
d(CGCm
8
GCG)
2
(
2
)
0.81
14.5 +- 1.2
46.2 +- 2.8
d(CGCGCG)
2
-0.80
11.8 +- 0.6
42.6 +- 2.2
a
Z-B conformational transition was analyzed by a two-state model from the data below 27oC. Thermodynamic parameters were obtained by plotting In(fraction
Z/fraction B) versus 1/T.
The chemical shifts of all resonances are tabulated in Table
1
. Note that all cytidine H
2'
and H
5'
resonances are unusually upfield (~1.7 and 2.6 p.p.m. respectively), analogous to those seen before (
6
,
19
). The upfield shifts are due to the orientation of the sugar moiety of the dC
nucleotide in Z-DNA, which places the H
2'
and H
5'
protons directly under the ring current of the neighboring 5'- and 3'-dG guanine bases respectively.
All our data point to the inevitable conclusion that d(CGC[m
8
G]CG)
2
has a structure consistent with Z-DNA. We constructed a model of d(CGC[m
8
G]CG)
2
by appropriate methylation of the Z-DNA d(CGCGCG)
2
crystal structure (
20
) and subjected it to a combined SPEDREF (
13
) and NOE-constrained refinement (
15
). We measured 710 NOE integrals as the input for the NOE-restrained refinement. The refined structure, which has an NMR
R
factor of 15.2%, is shown in Figure
3
. The NOE-refined structure is very similar to the d(CGCGCG)
2
Z-DNA structure determined by X-ray crystallography (
20
). The r.m.s. difference between the two structures is only 0.6 Å. The cytidine residues are in the
anti
/C2'-
endo
conformation, whereas the guanosine residues are in the
syn
/C3'-
endo
conformation (except for the 3'-terminal guanosines, which have a mixed C2'-
endo
/C3'-
endo
sugar pucker). In the m
8
G-modified Z-DNA structure the hydrophobic C
8
-methyl groups are located in the periphery of the helix and prominently
exposed to the solvent region. In contrast, in the m
5
C-modified Z-DNA structure the C
5
-methyl groups form hydrophobic patches in the small recessed area of the
concave `major groove' (Fig.
3
). The simulated NOESY spectra based on the refined model agree with the
observed data (Fig.
2
). To the best of our knowledge this is the first example of a refined structure
of Z-form DNA by NMR under physiological salt conditions without added organic
solvent or divalent cation.
Dynamics of Z-DNA
Z-DNA has been shown to have unusual rigidity (
2
). The measured T
1
relaxation inversion recovery time (T
1
IR) of 2.7 s for the d(CGC[m
8
G]CG)
2
helix supports this notion. For a B-DNA hexamer the averaged T
1
IR is ~1.7 s. The stiffness of the Z-DNA double helix is also reflected in the remarkably slow exchange
rate of its various exchangeable protons, including the G imino, G amino and C
amino protons. It has been shown that the G imino and C amino protons exchange
with water in 30 and 50 min respectively, whereas the G amino protons exchange
in 330 min, at 5oC and pH 7 (
21
). Our ability to obtain a stable Z-DNA structure under physiological conditions affords a unique opportunity
to investigate the behavior of the exchangeable protons.
Thermodynamic properties
The effect of m
8
G substitution on the thermodynamic stability of the Z conformation was examined
by measuring the proportions of the Z, B and SS forms at various temperatures.
Figure
1
B shows the CD spectra of d(CGC[m
8
G]CCG)
2
(
2
) in 2.6 M NaCl solution at various temperatures. At 2oC it is nearly 100% Z-DNA. The proportion of B increased with increasing temperature. For
comparison, d(CGCGCG)
2
under the same salt conditions consisted of a 1:1 mixture of B and Z. The
proportions of Z, B and SS for a m
8
G-containing oligomer were determined at various temperatures by means of CD
and UV spectroscopy as previously reported (
16
). A similar temperature-dependent B:Z equilibrium has also been observed for d(CGC[m
8
G]CCG)
2
at 30 mM salt concentration by NMR spectroscopy (Fig.
5
).
The results for
2
and d(CGCGCG) at 2.6 M NaCl as a function of temperature are shown in Figure
6
a and b. The thermodynamic parameters for the Z-B transition of the hexamer were determined from the data below 27oC, where >97% of the hexamers are in either the Z or B form. A van't
Hoff plot for the Z-B transition of d(CGCGCG)
2
and d(CGC[m
8
G]CG)
2
and the resulting [Delta]
H
and [Delta]
S
are shown in Figure
6
c and Table
2
respectively. It is evident that the large stabilization of the Z conformation
by introducing a methyl group at the guanine C8 position is enthalpic in origin
and that the methyl substitution stabilizes the Z form by at least 0.8
kcal/mol, which roughly corresponds to half of the reported free energy (~1-2 kcal/mol) (
22
-
24
) required to shift the equilibrium to the
syn
conformation of C
8
-substituted deoxyguanosines. A similar extent of stabilization of
syn
conformation by incorporating 8-bromodeoxyguanosine into oligomers has recently been reported for a G
quartet structure (
25
).
When A[middot]T base pairs are inserted into alternating (C[middot]G)
n
sequences the B-Z transition is known to become more difficult (
3
,
4
,
26
). For instance, the 5-methylcytosine (m
5
C)-containing octadecamer d(G-m
5
C)
4
A
Br
U(G-m
5
C)
4
(
3
) was found to retain the typical B form even at 4.0 M NaCl. Thus we examined
the properties of various types of m
8
G-containing oligomers having an A[middot]T base pair in order to evaluate Z form stabilization induced by
incorporation of m
8
G (Table
3
). In general the Z conformation was further stabilized by increasing the number
of m
8
G incorporated and destabilized by incorporating
syn
-A and
syn
-T. The CD spectrum of d(C[m
8
G]CAT[m
8
G]CG)
2
(
4
) indicates that this oligomer is converted from coil to Z with a mid-point at 45 mM NaCl. Oligomer
5
, which is obtained by replacing the terminal G[middot]C base pairs of
4
with A[middot]T base pairs, maintained the Z conformation with a mid-point at 470 mM NaCl. The incorporation of m
8
G into only one strand is also capable of stabilizing the Z conformation
considerably (oligomer
6
). A non-alternating pyrimidine-purine sequence has been shown to destabilize the Z conformation
due to the energetically disfavored
syn
conformation of pyrimidine nucleosides (
3
,
4
). One of the central G[middot]C base pairs of d(CGCGCGCG)
2
can be replaced by a T[middot]A base pair without significantly increasing the mid-point NaCl concentration, if the duplex incorporates two m
8
G into each strand (oligomer
7
). Such a low salt concentration requirement of 120 mM for an imperfect Z-DNA (out-of-alternation pyrimidine-purine sequence) is remarkable. Our results suggest
that we can now study many heretofore inaccessible DNA conformations involving
Z-DNA, e.g. the B-Z junction and the Z-Z junction. Such experiments are under way.
Conclusion
The substitution of a methyl group at the guanine C
8
position dramatically stabilizes the Z conformation of short oligonucleotides with a variety of base sequences. Some of these m
8
G-modified oligomers exist as a stable Z form under physiological salt
conditions without added organic solvent or divalent metal (
3
-
7
). While significant information on specific chemical reactions for DNA local
structures has been accumulated during the past several years (
27
-
30
), considerably less is known about the origin of these specificities.
Incorporation of the m
8
G moiety into DNA oligomers could be a powerful tool to examine the molecular
basis for many types of Z conformation-specific reactions at the oligomer level under physiological conditions.
ACKNOWLEDGEMENTS
The Kyoto part of this work was supported by a Grant-in-Aid for Priority Research from the Ministry of Education and the
Research Foundation for Opto-Science and Technology and the Urbana part was supported by NIH grant GM-41612 to AH-JW.
REFERENCES
1 Cozzarelli,N.R. and Wang,J.C. (1990) DNA Topology and Its Biological Effects. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
2 Sinden,R.R. (1994) DNA Structure and Function. Academic Press, New York, NY.
3 Rich,A., Nordheim,A. and Wang,A.H.-J. (1984) Annu. Rev. Biochem., 53,791-846.
